Gene therapy is a general term that indicates a technique to treat disease by replacing an abnormal gene that causes disease in cells or tissues in a patient with a target gene or by inserting a target gene that is helpful for the treatment of disease therein. In the early development days of gene therapy products, the major principle of gene therapy was to induce the specific gene expression by inserting foreign DNA into the target cell chromosome. However, today's gene therapy includes the method using antisense to inhibit the expression of a gene related to a specific disease by using antisense oligodeoxynucleotide or siRNA.
The mentioned gene therapy takes a totally different approach from the conventional treatment methods, so that can investigate a reason of disease at a molecular level for better treatment. This method is also advantageous in reducing unnecessary side effects frequently observed in other treatment methods due to its nucleotide sequence specific function that can eliminate the factors related to major diseases. Such a method targeting gene as the above does not need a separate step of optimization in the course of drug production as long as the nucleotide sequence of a target gene for the control of expression level is identified, indicating the production procedure is simpler than that for an antibody or compound drugs. In addition, this method can target any disease which other methods cannot take it as a target, once a causing gene of the disease is identified, suggesting that gene therapy has a full potential as a next generation treatment agent. Numbers of researches confirmed that the chances of successful treatment for incurable disease, cancer, AIDS, genetic disorder, and neurologic disorder which are hard to treat with the conventional medicinal treatment methods could be increased with gene therapy, and therefore gene therapy is now being applied to actual clinical trials (YOUNG et al, 2006).
A gene medicine is composed of a gene transfer vector and a therapeutic gene. As a tool to deliver a gene in vivo, the gene transfer vector is largely divided into a viral vector and a non-viral vector. The viral vector is prepared by making a virus non-replicable by eliminating most of or a part of essential genes and instead inserting a therapeutic gene therein (Lotze M T et al., Cancer Gene Therapy, 9:692-699, 2002). The viral vector can deliver a gene with high efficiency but has problems of difficulty in mass-production according to the types of virus, causing immune response, toxicity, or introducing replicable virus, etc. The major viral vectors being used for the development of a gene medicine are exemplified by retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, and pox virus etc. In the meantime, the non-viral vector does not cause immune response, has low toxicity, and is easy to mass-produce, but is not efficient in delivering a gene and can only induce temporary gene expression.
One of the most frequently used viral vector in the field of clinical trial is a retrovirus vector, which was first used in the first gene therapy clinical trial performed by NIH in 1990. This vector is regarded as the most useful vector for a stable gene insertion. The retrovirus vector based on moloney murine leukemia virus (MoMLV) is widely used in various clinical trials for gene therapy.
The non-replicating retrovirus whose self-replication is defective is suitable for the insertion of a relatively big gene. The titer thereof is 106˜107 pfu/ml, so there is not a big problem in the infection of a target cell with this vector. Also, the packaging cell line has been already established, indicating that the preparation of this vector is easy. In addition, the retrovirus vector can be scaled-up by means of inserting a therapeutic gene in the retrovirus plasmid, with which the packaging cell line is infected to produce the recombinant virus; and infecting a target cell with the produced recombinant virus. However, there is a chance of mutation caused in the course of retroviral integration into chromosome.
Meanwhile, the genome stability of the self-replicable retrovirus vector in the proliferating cell such as a cancer cell has been an issue. Besides, a foreign gene that can be inserted in the self-replicable virus vector for gene therapy is limited up to 1.3 kb in the size, indicating the insertion of various therapeutic genes is not easy (J. of virology, Vol. 75, 6989-6998, 2001).
The therapeutic gene usable for gene therapy is exemplified by the gene inducing cancer cell apoptosis by using a prodrug such as herpes simplex virus thymidine kinase or cytosine deaminase; the cytokine gene accelerating immune response such as interleukin-12 or GM-CSF, etc; and the tumor specific antigen gene such as CEA or Her-2, etc. (Gottesman M M, Cancer Gene Therapy, 10:501-508, 2003). Suicide gene kills cancer cells when it is delivered into the cancer cells. Cytokine gene or tumor specific antigen gene attacks cancer cells by activating immune response against cancer.
Recently, studies have been actively going on about the synthesis technique of enzyme/prodrug that exhibits selective antitumor effect on malignant tumor. Realistically, when suicide gene is expressed in cancer tissues and its precursor is administered in vivo systemically, it does not cause toxicity in normal cells but the precursor is converted into a toxic material to kill tumor cells only in those tumor cells where the therapeutic gene is expressed.
One of the most generally used suicide gene is herpes simplex virus thymidine kinase (HSV-TK). When the prodrug ganciclovir (GCV) which does no harm on cells is converted into a cytotoxic material through enzyme reaction, it acquires bystander effect that can induce the apoptosis of not only the cells harboring the suicide gene but also the neighboring cells by gap junction. The effect and stability of the gene has been confirmed so far until the phase 3 clinical trial (human gene therapy, 4:725-731, 1993; molecular therapy, 1:195-203, 2000).
Another suicide gene is cytosine deaminase (CD), which induces deamination of 5-fluorocytosine (5-FC) to produce a powerful anticancer agent 5-fluorouracil (5-FU). 5-FU is metabolized into 5-fluorouridine triphosphate (5-FUTP) and 5-fluorodeoxyuridine monophosphate (5-FdUMP). 5-FUTP fused to ribonucleic acid interrupts the synthesis of ribosomal ribonucleic acid and messenger ribonucleic acid. In the meantime, 5-FdUMP suppresses thymidine synthase irreversibly, leading to the inhibition of DNA synthesis. Therefore, tumor cells can be selectively killed by converting such prodrugs as GCV and 5-FC into toxic metabolites in the tumor cells expressing TK or CD.
Using more than 2 different therapeutic genes simultaneously for gene therapy is more efficient in disease treatment and particularly in dealing with the disease displaying resistance against a specific gene used for gene therapy. In relation to such a technique, a gene therapy vector system that can express TK and CD simultaneously in the cancer tissue is very much advantageous, particularly for the treatment of such cancers reported to have resistance against TK and CD treatment. However, when HSV-TK and CD are both inserted in a replicating-retrovirus vector (RRV), the genome size becomes 10 kb or more, indicating the insertion of the two genes at the same time in a single retrovirus vector is in fact impossible. When a replicating-retrovirus vector is used for gene therapy, it has to contain a foreign gene in addition to its original endogenous genomic RNA, so that the size of the genomic RNA is getting bigger and has a high potential of therapeutic gene loss because of recombination caused by the additional heterologous nucleotide sequence introduced therein, making the construction of a vector system difficult.
The present inventors tried to develop a safe virus vector system for gene therapy which is free from recombination. As a result, the inventors developed a TK/CD combined self-replicating retrovirus vector system containing both HSV-TK and CD genes with excellent stability but without worry of recombination caused gene loss. The present inventors confirmed that the said vector could induce cancer cell death by using the prodrug GCV or 5-FC and be selectively applied to the treatment of specific cancer that showed resistance against either TK or CD by selecting a proper therapeutic gene and the prodrug thereof. The present inventors further completed this invention by confirming that the said vector thereby could be useful as a pharmaceutical composition for the prevention or treatment of cancer.